A ligand discovery toolbox for the WWE domain family of human E3 ligases

The WWE domain is a relatively under-researched domain found in twelve human proteins and characterized by a conserved tryptophan-tryptophan-glutamate (WWE) sequence motif. Six of these WWE domain-containing proteins also contain domains with E3 ubiquitin ligase activity. The general recognition of poly-ADP-ribosylated substrates by WWE domains suggests a potential avenue for development of Proteolysis-Targeting Chimeras (PROTACs). Here, we present novel crystal structures of the HUWE1, TRIP12, and DTX1 WWE domains in complex with PAR building blocks and their analogs, thus enabling a comprehensive analysis of the PAR binding site structural diversity. Furthermore, we introduce a versatile toolbox of biophysical and biochemical assays for the discovery and characterization of novel WWE domain binders, including fluorescence polarization-based PAR binding and displacement assays, 15N-NMR-based binding affinity assays and 19F-NMR-based competition assays. Through these assays, we have characterized the binding of monomeric iso-ADP-ribose (iso-ADPr) and its nucleotide analogs with the aforementioned WWE proteins. Finally, we have utilized the assay toolbox to screen a small molecule fragment library leading to the successful discovery of novel ligands targeting the HUWE1 WWE domain.


Figure S1 .
Figure S1.The 28 amino acids that are missing in isoform 1 of the TRIP12 WWE domain.(a) Sequence alignment of the WWE domains of TRIP12 isoforms 1 and 2 with the HUWE1 WWE domain sequence.The secondary structure elements are annotated based on the HUWE1 WWE apo structure (PDB ID: 6MIW).Residues that form β-strands 3 and 4 are missing in isoform 1 of the TRIP12 WWE domain sequence.(b) AlphaFold predicted structure of residues 749-836 that are annotated to form the WWE domain in the isoform 1 TRIP12 sequence.(c) AlphaFold predicted structure of TRIP12 isoform 2 WWE domain.(d) An overlay of the HUWE1 WWE apo structure (PDB ID: 6MIW) and the AlphaFold predicted structures of the isoform 1 (green) and isoform 2 (slate blue) TRIP12 WWE domains.Due to the missing 28-aa segment, the isoform 1 TRIP12 WWE structural fold is altered.In addition, the segment rendered in yellow in the isoform 1 TRIP12 WWE predicted structure (panel A) is not part of the WWE domain despite being annotated so.

Figure S2 :
Figure S2: Domain architecture of Deltex E3 ligases containing a WWE domain.(a) Domain organization of human and fruit fly DTX E3 ligases with cutoffs defined by the NCBI conserved domains database.Domains are drawn to scale and are defined as follows: WWE1/2 = first/second WWE domain, RING-H2 = Really Interesting New Gene finger domain, DTC = Deltex C-terminal domain.(b) Phylogenetic tree and complete sequence alignment of human DTX1, DTX2 and DTX4 WWE domains with the Drosophila Deltex WWE domain.The residues have been re-numbered starting from one and the phylogenetic tree was constructed without considering branch lengths.

Figure S3 :
Figure S3: 15 N-HSQC NMR based binding studies of ATP-analogues.(a,b,e) 15 N-HSQC spectra of (a) DTX2, (b) RNF146, and (e) TRIP12 overlaid with protein in the presence of 1 mM 2F-ATP (1).(c, d, f) 15 N-HSQC NMR Kd titration assay and zoom-in on a peak upon a twofold titration of 2F-ATP (1) to (c) DTX2, (d) RNF146, and (f) TRIP12 using a concentration range of 62.5 µM to 2 mM.NMR Kd values originate from distinct samples (n=1) measured for each concentration, mean Kds obtained from curves of selected cross peaks ± standard deviations.For the titration curve the ligand concentrations are plotted on the x-axis and the Δδ chemical shifts on the y-axis.

Figure S4 :
Figure S4: 15 N-HSQC-NMR titration curves of HUWE1 and RNF146.(a-f) For the titration curve the ligand concentrations are plotted on the x-axis and the Δδ chemical shifts on the y-axis.The maximal plotted concentrations are up to compound saturation.The Δδ chemical shifts of one to four different peaks are followed of a single titration experiment.The position of the individual peak on the 1 H-and 15 N-scale of a 1 H-15 N-HSQC spectrum is labeled in ppms in the legend.Below the titration curve is a table with the position of the peaks in the column headers and the protein concentration in the experiment (Ptot), the dissociation constant (Kd) in µM and the maximum specific binding (Bmax) of each of the analyzed peaks in the rows.The titration curve of (a) iso-ADPr, (b) ADPr, (c) ATP, (d) 2F-ATP (1), (e) 2'F-ATP (2) to HUWE1 and (f) iso-ADPr to RNF146 is plotted..NMR Kd values originate from distinct samples (n=1) measured for each concentration, mean Kds obtained from curves of selected cross peaks ± standard deviations

Figure S5 :
Figure S5: 15N-HSQC-NMR titration curves of RNF146 and TRIP12-2.(a-f) For the titration curve the ligand concentrations are plotted on the x-axis and the Δδ chemical shifts on the y-axis.The maximal plotted concentrations are up to compound saturation.The Δδ chemical shifts of one to four different peaks are followed of a single titration experiment.The position of the individual peak on the 1 H-and 15 N-scale of a 1 H-15 N-HSQC spectrum is labeled in ppms in the legend.Below the titration curve is a table with the position of the peaks in the column headers and the protein concentration in the experiment (Ptot), the dissociation constant (Kd) in µM and the maximum specific binding (Bmax) of each of the analyzed peaks in the rows.The titration curve of (a) ADPr, (b) ATP (c) 2F-ATP (1), (d) 2'F-ATP (2) to RNF146 and (e) iso-ADPr and (f) ADPr to RNF146 is plotted.NMR Kd values originate from distinct samples (n=1) measured for each concentration, mean Kds obtained from curves of selected cross peaks ± standard deviations.

Figure S6 :
Figure S6: 15 N-HSQC-NMR titration curves of TRIP12-2 and DTX1.(a-b) For the titration curve the ligand concentrations are plotted on the x-axis and the Δδ chemical shifts on the y-axis.The maximal plotted concentrations are up to compound saturation.The Δδ chemical shifts of one to four different peaks are followed of a single titration experiment.The position of the individual peak on the 1 H-and 15 N-scale of a 1 H-15 N-HSQC spectrum is labeled in ppms in the legend.Below the titration curve is a table with the position of the peaks in the column headers and the protein concentration in the experiment (Ptot), the dissociation constant (Kd) in µM and the maximum specific binding (Bmax) of each of the analyzed peaks in the rows.The titration curve of (a) ATP, (b) 2F-ATP (1), (c) 2'F-ATP (2) to TRIP12-2 and (d) iso-ADPr, (e) ADPr and (f) ATP to DTX1 is plotted.NMR Kd values originate from distinct samples (n=1) measured for each concentration, mean Kds obtained from curves of selected cross peaks ± standard deviations

Figure S7 :
Figure S7: 15 N-HSQC-NMR titration curves of DTX1 and DTX2.(a-f) For the titration curve the ligand concentrations are plotted on the x-axis and the Δδ chemical shifts on the y-axis.The maximal plotted concentrations are up to compound saturation.The Δδ chemical shifts of one to four different peaks are followed of a single titration experiment.The position of the individual peak on the 1 H-and 15 N-scale of a 1 H-15 N-HSQC spectrum is labeled in ppms in the legend.Below the titration curve is a table with the position of the peaks in the column headers and the protein concentration in the experiment (Ptot), the dissociation constant (Kd) in µM and the maximum specific binding (Bmax) of each of the analyzed peaks in the rows.The titration curve of (a) 2F-ATP (1) and (b) 2'F-ATP (2) to DTX1 and (c) ADPr, (d) ATP, (e) 2F-ATP (1) and (f) 2'F-ADP (2) to DTX2 is plotted.NMR Kd values originate from distinct samples (n=1) measured for each concentration, mean K d s obtained from curves of selected cross peaks ± standard deviations.

Figure S9 :
Figure S9: Fo − Fc difference density (green mesh) calculated when the ligands of interest are omitted (contoured at 3σ except for DTX1-WWE1/2-ADP structures which are shown at a contour level of 2σ due to lower resolution of the respective structures).

Figure S10 :
Figure S10: The fold conservation in the Deltex tandem WWE domains.A superposition of the reported Drosophila Deltex structure (2A90; cyan), the DTX1-WWE1-ADP structure (grey) and the DTX1-WWE2-ADP (yellow) showing fold conservation of both the WWE domains as well as the linking region.The ADP molecules (labeled) are rendered as sticks.

Figure S11 :
Figure S11: Comparisons of the natural WWE ligands binding sites.(a) The binding site of the iso-ADPr bound RNF146 WWE domain (PDB: 3V3L).Iso-ADPr is shown as orange sticks and potential hydrogen bonds are shown as black dashes.(b) Superposition of the HUWE1-ADPr WWE domain (magenta) with iso-ADPr bound RNF146 WWE domain (green; PDB: 3V3L), showing the differences in the binding mode of the two ligands as well as the residues that interact with each ligand to confer specificity.ADPr and iso-ADPr are depicted as grey and orange thin sticks, respectively.The conserved residues in the binding site are shown as lines while the non-conserved ones are shown as sticks.The binding site residues in the HUWE1-ADPr structure are labeled and possible interactions involving RNF146 and iso-ADPr are shown as black dashes.(c) Superposition of the TRIP12-ADP WWE domain (slate blue) with the RNF146-iso-ADPr WWE domain (green).ADP and iso-ADPr are rendered as cyan and orange thin sticks respectively.Conserved residues in the vicinity of the ligands are shown as lines while non-conserved ligand-interacting residues are shown as sticks.The binding site residues in the TRIP12-ADP structure are labeled and the possible interactions of RNF146 with iso-ADPr are shown as black dashes.(d) A superposition of ADPr-bound HUWE1 WWE domain (magenta) and ADP-bound TRIP12 WWE domain (slate blue).ADPr is shown as grey lines, ADP as cyan lines, the conserved ligand-interacting residues as lines and the non-conserved residues as sticks.

Figure S12 :
Figure S12: FP-based PAR11-mer direct binding to the HUWE1 wildtype (HUWE1-WT) and HUWE1 N1674T mutant (HUWE1-N1674T) WWE domains.The fluorescence polarization percentage (% FP) of the reference is plotted as a function of HUWE1 protein concentration in μM using the logarithmic scale.The experiments were conducted in distinct triplicates (n=3) in 1 experimental repeat.The plotted values represent the averages from the 3 replicates.

Figure S13 :
Figure S13: Comparison of the ADP-bound DTX1 WWE domains.A superposition of ADP bound DTX1 WWE1 (DTX1-WWE1-ADP, grey) and WWE2 (DTX1-WWE2-ADP, yellow) domains, highlighting amino acid differences in the binding sites between the two domains and the binding mode of the bound ADP molecules.ADP in the WWE1 domain is shown as cyan lines while the one in the WWE2 domain is shown as magenta lines.The conserved residues between the two binding sites are rendered as lines, while the non-conserved ones are depicted as sticks.

Figure S14 :
Figure S14: Comparisons of the ligand binding sites of RNF146-iso-ADPr and ADP-bound DTX1 WWE domains.(a) A superposition of the iso-ADPr bound RNF146 WWE domain (green; PDB: 3V3L) and the ADP bound DTX1 WWE1 domain (grey; DTX1-WWE1-ADP).Iso-ADPr is depicted as thin orange sticks while ADP is shown as thin cyan sticks.The conserved residues in the binding site are shown as lines while the non-conserved ones are shown as sticks.Residues of the DTX1-WWE1-ADP WWE domain are labeled and possible interactions involving RNF146 and iso-ADPr are shown as black dashes.(b) A superposition of the same structures in panel (a) showing the differences in the binding site of the distal ribose and phosphate groups of iso-ADPr.The binding site residues of both structures are labeled (RNF146-iso-ADPr numbers are in brackets) and possible interactions of iso-ADPr with RNF146 are shown as black dashes.(c) A superposition of the ADP bound WWE2 domain of DTX1-WWE2-ADP (yellow) and the iso-ADPr bound RNF146 WWE domain structure (green).The conserved residues in the binding site are shown as lines while the non-conserved ones are shown as sticks.Residues of the DTX1-WWE2-ADP WWE domain are labeled and possible interactions involving RNF146 and iso-ADPr are shown as black dashes.(d) A zoomed-in image of the the binding site of the distal ribose and phosphate groups of iso-ADPr in the superimposed structures in panel (c).The binding site residues of both structures are labeled (RNF146-iso-ADPr numbers are in brackets) and possible interactions of iso-ADPr with RNF146 are shown as black dashes.

Figure S15 :
Figure S15: The AlphaFold predicted structure of the human DTX2 tandem WWE domains.(a) The predicted fold of the tandem DTX2 WWE domains (green) compared to the DTX1-WWE1-ADP complex structure with the ADP molecule (grey sticks) in the WWE1 domain (grey cartoons).(b) The binding site of the predicted DTX2 WWE1 structure (green) compared to that of the WWE1 domain (grey) in complex with ADP (grey lines) in the DTX1-WWE1-ADP structure.All the conserved residues in the DTX1 WWE1 binding site (sticks) are similarly positioned in the DTX2 WWE1 domain (also in sticks).Non-conserved residues are shown as lines and DTX2 WWE1 residues are labeled.(c)The binding site of the predicted DTX2 WWE2 domain (green) compared to that of the WWE2 domain (yellow) in complex with ADP (yellow sticks) in the DTX1-WWE2-ADP structure.The DTX2 WWE2 domain residues are labeled, conserved residues are shown as sticks and non-conserved residues are depicted as lines.

Figure S16 :
Figure S16: The binding of ATP to TRIP12 and DTX1 WWE domains.(a) The binding site of the TRIP12-ATP structure showing the binding mode and interactions of ATP (yellow sticks) with the protein.(b) A superposition of the TRIP12-ATP (green) with the TRIP12-ADP (slate blue) structures, showing the conservation of sidechain positions and orientation in the binding sites, except for Arg807 sidechain, which moves to interact with the terminal phosphate group of ATP.ADP in the TRIP12-ADP structure is depicted as cyan sticks, while ATP in the TRIP12-ATP structure is shown as yellow sticks.Potential hydrogen bonds are shown as dashes.(c) The binding site of the DTX1-WWE1-ATP structure showing the binding mode and interactions of ATP (yellow sticks) with the protein.(d) A superposition of the ATP bound DTX1 WWE1 (brown) and the ADP-bound DTX1 WWE1 domain (grey), showing the conservation of the sidechain positions despite the slight differences in the conformation of the ligands.ATP in the DTX1-WWE1-ATP structure is shown as yellow sticks while ADP in the DTX1-WWE1-ADP structure is depicted as cyan sticks.Potential hydrogen bonds are shown as dashes.

Figure S17 :
Figure S17: Comparisons of ATP binding to RNF146, TRIP12 and DTX1 WWE domains.(a) The binding site of the mouse RNF146-ATP NMR structure (PDB: 2RSF; ensemble 1) showing the binding mode and interactions of ATP (yellow sticks) with the protein.Potential hydrogen bonds are shown as black dashes.(b) A superposition of the RNF146-ATP (ensemble 1, slate) and the TRIP12-ATP (green) structures, highlighting the similarities and differences in the ATP binding mode.The ATP molecules are depicted as thin sticks in the same color as the respective proteins.Conserved residues are shown as lines, while the non-conserved ones are rendered as thick sticks.(c) A superposition of the ATP bound mouse RNF146 WWE domain (slate; ensemble 1) and the ATP bound DTX1 WWE1 domain (brown) structures.Conserved residues are rendered as lines, while non-conserved residues are shown as thick sticks.The ATP molecules are depicted as thin sticks in the same color as the respective proteins.

Table S2 :
RMSD values for pairwise alignment of the reported structure of RNF146 WWE with iso-ADPr (PDB ID: 3V3L) against the nine structures reported in this study.